Use of Synthetic Biology in the Development of Bacterial Adhesins for Skin Grafting applications

Lead Research Organisation: University of Sheffield
Department Name: Clinical Dentistry

Abstract

Despite recent medical advances in the field of skin grafting there are still a large number of skin graft treatments that are unsuccessful. This failure of grafts is largely due to poor 'sticking' to the damaged area to be treated, commonly termed the wound bed. One of the reasons these 'bad' wound beds are difficult to adhere to is due to the lack of naturally occurring sticky molecules. Therefore one approach might be to use a glue that introduces some of these proteins into the wound or that can bind strongly to the few that are present and form a bond between incoming grafted cells and the wound bed to be treated. One of the proteins that is present in the bad wound beds is a molecule called Type 1 collagen.

In this project we plan to use a novel approach to produce a 'bioglue' that will promote sticking of skin grafts to bad wound beds via type 1 collagen. This project will take advantage of the fact that several human infecting bacteria produce small molecules that bind strongly to collagen 1 which they use to stick down to human cell surfaces such as the airways or skin. Our idea is to engineer a selection of these small protein molecules into a protein fibre- also produced by bacteria, called the flagellum.

The approach to engineering these protein fibres will follow the philosophy of an emerging biological field known as synthetic biology. At its core synthetic biology aims to design or re-engineer biological parts into often novel combinations in a modular manner that allows easy re-use and re-engineering of these parts for use in alternative applications. One can consider an analogy with Lego in which the same component parts can be used to produce a car, a house or a crane. To this end the bacteria flagellum is amenable to this kind of manipulation. The sections of this protein that direct formation of a fibre form can be thought of as two modules at each end of the protein which in isolation would form a fibre forming protein device with a central section that can be replaced with another module. We have engineered such a device to accept sticky protein modules in place of this section and so display thousands of these sticky proteins along the length of the fibre. In our initial studies we have placed one of these type 1 collagen molecules and shown that it can bind type 1 collagen and improve sticking of skin-like cells in a laboratory setting.

The aim of this project is to produce a range of these type 1 collagen binding sticky flagella and test their ability to stick natural skin cells to surfaces and also improve adherence of these cells in a laboratory based skin grafting assay that we have developed.
As well as applications to skin grafting these bioglues may also have other medical applications in situations where improving adherence of human tissue is required. In addition these easy to manipulate flagella display devices will be of great use to this emerging synthetic biology community for a range of future applications.

Technical Summary

Since the 1980's cultured tissue engineered keratinocytes have been used to treat burns and chronic wounds. However, cultured cells usually fail to attach well to challenging wound beds, as are often present in burns or chronic non-healing ulcer patients. These wound beds are characterised by poor vasculature and a dearth of extracellular matrix proteins that ensure attachment of the epidermis to dermis (i.e. Collagen IV, VII and laminin). They contain predominantly Collagen I, are rich in degradative enzymes, sometimes infected and usually poorly vascularised. Even conventional split-thickness skin grafts can fail to attach well on such wound beds.
We aim to produce a 'bioglue' targeted at attaching split thickness skin grafts or cultured cells to collagen I. This bioglue must increase adhesion in these situations and persist in the body long enough (5-10 days) for adhered skin cells to begin remodelling wound beds, promote vasculature and ultimately restore barrier function.
Our approach is to use a synthetic biology pathway to redesign the bacterial flagellum to produce adhesive protein fibres. We have redeveloped the flagellin molecule into a modular adhesin presentation device comprising the D0 and D1 polymerisation domains but lacking the central domains D2 and D3. This device has been engineered to accept a range of adhesin domains from bacteria and humans in a modular fashion via restriction sites. After biochemical characterisation, these will be introduced into our poor wound bed model, lacking basement membrane and fibroblasts and growth/ adhesion of cells assessed. We have engineered a prototype flagellin device to carry a module comprising the cna adhesin from Staphylococcus aureus and shown that it binds collagen I and promotes adhesion of a Skin cancer cell line to tissue culture plastic. In this proposal we will take to the next stage of testing and improve both yield, purity and efficacy while also reducing any potential immunogenicity.

Planned Impact

This is an interdisciplinary application, located at the interface between biological science and (tissue) engineering. While this is a pilot/proof of principle type project of only 12 months duration it may have a number of potential impacts in the longer term.
Economic and societal impacts: While it is difficult to estimate how much the NHS spends on skin grafting applications it has been estimated that the UK alone spends £3 billion per year on the management of chronic wounds. In addition there are an estimated 175,000 A and E visits are due to burn injuries and of those over 13,000 are admitted. Many of the problems come from the recalcitrant nature of grafting onto challenging or non-ideal wound beds, i.e. those with a dearth of basement membrane or adhesive molecules. Treatment of burns is a priority of the NHS with the inception in 2008 of regional burn centres to streamline and improve the standard of treatment in this clinical sphere. Any improvement on the currently used methods for skin grafting onto challenging wound beds would have a profound effect and impact an a large number of patients in the UK and potentially further afield.
Major beneficiaries will include academics who study wound healing, skin biology, tissue engineering, microbiology and most importantly clinical practice in the long run. This would also be an exemplar project in the field of synthetic biology in the UK, which has to date much promise but has not yet delivered on this potential. In addition if the novel adhesives to be investigated in this project prove successful there will be obvious commercial potential in the field of surgical materials. In addition there will clearly be a number of other clinical applications for this technology, such as in the improvement of gingival implantation, an area of investigation in the dental school in Sheffield, and as a general adhesive in a number of clinical spheres.
Research staff: Development of these adhesives will require reserch staff that will benefit by working collaboratively with researchers from a number of different fields across tissue engineering, microbiology and biological engineering. The project will provide opportunities for publishing in new scientific areas, presenting to diverse audiences and strengthening the direction of their careers. The PDRAs will benefit by publishing in high-impact journals and will be encouraged to be active in the preparation of subsequent funding applications in this area of research. In addition Dr Stafford, Prof MacNeil and Prof Wright are attempting, on the back of the MATEs network activity, to build up a critical mass of researchers in the area of Synthetic Biology with a focus on applications based research that will impact the area of tissue engineering chiefly. Thus any investment will have a great impact on the face of interdisciplinary research in Sheffield and the UK.
Policy regulators: If a novel, biocompatible skin graft adhesive were produced this could potentially change the face of decision making in the treatment of burns victims or those with chronic wounds and be considered in documents such as burn care reviews etc.
Industry: It is intended that the development of a novel skin graft adhesin will be translated to industry. Prof MacNeil has strong links with a number of possible partners including Smith and Nephew, with whom she is developing a 3D model for infection, amoung others. Such a programme of activity will place the UK at the forefront of novel clinical adhesives and will be a significant advance for the UK to lead this field with associated economic benefits.

Publications

10 25 50
 
Description The aim of the project was the production of chimeric synthetic flagellin proteins containing potential bioglue domains by the model bacterium E.coli with the aim of aiding in tissue engineering applications to aid skin graft success. The project succeeded in scaling up and standardising production of the synthetic chimeric bioglue proteins and showed in model lab systems that they bind the target human proteins showing these molecules have potential for use in the adhesion of skin cells to surfaces that might ultimately have applications in skin grafting or implantation of cultured primary skin cells into 'bad' would beds.

We have also been addressing the issue of improving production of the chimeric flagella proteins (and other biotechnological important proteins in E. coli), a task that has involved performing a quantitative proteomic assessment of global protein expression during expression. We are currently analysing these data and will use this information to forward engineer the strains to produce more protein- data that will improve understanding of production of proteins in E.coli and aid our efforts in collaborations with biotechnology companies. This data willl support a new collaboration with Fujifilm Diosynth aimed at adressing this issue in an industrial biotechnology situation and which is funded at PoC level by the BBSRC NIIB CBMNet which we are now discussing with Fuji-Db (but with the second replacement for Bo Kara-namely Ian Hodgson).

This project now has a PhD student on an Indian Government studentship carrying on the work analysing the proteome dataset and implementing some of the idea generated by that work.

2016-17

This work had lead to further NIBB funding to carry examine flagellar secretion for biotech proteins- suppoted by CBMnet Biv, and to which Fuji contrinbuted £10,000 in kind.
Exploitation Route In the longer term the outcomes of this work have the potential to help in the treatment of patients in the NHS and for the production of novel bioglues by the biotech/ healthcare sectors.
While this technology is early stage we are well placed to exploit both the flagellin bioglues (via contact with Smith and Nephew- MacNeil) and secretion system (through Lonza- Wright).

In addition our dataset on the proteome of proteins expressing large amounts of flagellin will reveal bottlenecks in the proteome that can be engineered to improve production of flagella chimeras and other proteins to aid in our work on protein secretion and production that has now moved on to a collaborative effort between Sheffield (Stafford, Wright, Dobson), Birmingham (Henderson) and Fujifilm Diosynth (Dr Bo Kara initially and now Ian Hodgson- the third change in this role during the last year). These data will thus have direct impact on the UK Biotech sector to potentially produce novel IP for this area of the economy. We are currently writing a paper that this award has contributed to focussing on flagella secretion.
Sectors Healthcare,Manufacturing, including Industrial Biotechology

URL http://www.synbio.group.shef.ac.uk/synbio/news/bbsrcadhesins
 
Description CBMNet Business Innovation Voucher- with Fujifilm diosynth biotech
Amount £10,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Department Networks in Industrial Biotechnology and Bioenergy (NIBB)
Sector Academic/University
Country United Kingdom
Start 11/2016 
End 08/2017
 
Description PoC funding from BBSRC network in industrial biotechonology> CBMNet
Amount £48,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Department Networks in Industrial Biotechnology and Bioenergy (NIBB)
Sector Academic/University
Country United Kingdom
Start 02/2015 
End 07/2015
 
Description SynbiCITE - an Imperial College led Innovation and Knowledge Centre (IKC) in Synthetic Biology
Amount £5,074,190 (GBP)
Funding ID EP/L011573/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 10/2013 
End 10/2018
 
Description Discovery night 2013, University of Sheffield 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Dr Stafford and team, including staff employed on this grant, manned a stand at the Sheffield Discovery night where Dr Stafford's group presented an interactive display explaining various aspects of microbiological and synthetic biology research in the dental school. This included a prize competition to 'build a bacterium' where aspects of flagella production and motility were discussed- link: . In addition Dr S gave a public lecture entitled: 'the animalcules within: mini-creatures in your mouth'.

no actual impacts realised to date
Year(s) Of Engagement Activity 2013
URL http://www.sheffield.ac.uk/dentalschool/research/discoverynight/winnervisithttp://www.sheffield.ac.u...
 
Description iGEM 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact The team presented their work at the iGEM meeting, sparking several discusssions and providing great experience for the 8 undergraduates participating plus the 4 phD advisors and 3 instructors. They gained a Gold medal award at the giant jamboree and also engaged local schools,, businesses and regulatory authorities to discuss the impact and social and economic context of their project.

All 8 undergraduates have had a great experience and have gained training, international exposure and vital skills in both scientific but also social contextualising of their work. This goes beyond public engagement as they were mentored by social science colleagues to fully engage in the socio-economic impacts of their work. They also produced a wiki- below, with all the information on the project.
Year(s) Of Engagement Activity 2014
URL http://2014.igem.org/Team:Sheffield